Piper betle Phytochemical Analysis and Cytotoxicity Evaluation of Combined Bioactive Glass – P. betle on Dental Pulp Stem Cells

Objectives: Recent advances in endodontics emphasize regenerative strategies, utilizing both synthetic and natural bioactive materials to protect and regenerate the dentine-pulp complex. Piper betle (PB), a member of the Piperaceae family, possesses various medicinal properties. This study aims to analyze the phytochemical compounds of PB extract, evaluate the cytotoxicity of PB-conditioned media both individually and in combination with bioactive glass (BG) 45S5 Bioglass ® , and identify the chemical compounds present in optimal doses of PB and BG-conditioned media, separately and in combination (bioactive glass-piper betle [BGPB]). Material and Methods: The PB extract was prepared by ethanol maceration and analyzed using gas chromatography–mass spectrometry (GCMS) analysis to identify bioactive phytochemicals present. Three test groups were utilized (PB, BG, and BGPB-conditioned medium). Each test group’s conditioned medium was prepared at various concentrations through serial dilution. Cytotoxicity was assessed using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay on dental pulp stem cells (DPSC) on days 1, 2, 4, 7, and 14. The optimal concentration dose of each test group underwent GCMS analysis. Data were analyzed using repeated-measures analysis of variance with Bonferroni post hoc tests (Statistical Package for the Social Sciences v27; P ≤ 0.05). Results: The GCMS analysis of the PB extract revealed the presence of phenols, esters, ketones, carbohydrates, and nucleotides. DPSCs exposed to 4.00 mg/mL BG-conditioned media showed the highest cell viability, while cells cultured in PB-conditioned media exhibited the highest cell viability at a concentration of 31.25 µg/mL. Hence, these concentrations of PB and BG were selected for combination in further analysis. The BGPB-conditioned media showed the highest percentage of viable cells at a concentration of 4 mg/30 µg. Overall, the BGPB group demonstrated the highest cell viability, followed by the BG and PB groups. No significant difference was observed between the BGPB group and the BG group, but significant differences were found when comparing the BGPB group to the PB group. However, no significant difference in cell viability was detected between the BG and PB groups. Various active compounds, including phenolic compounds, terpenoids, esters, fatty acids, ketones, furans, imines, siloxanes, and amides, were detected in BG, PB, and BGPB-conditioned media. Conclusion: Within the limitations of this study, BGPB has shown potential to enhance the proliferation of DPSC in vitro at specific dosages and warrants further investigation for its regenerative properties.